Cardiovascular disease is the number one killer in the US. The incidence and severity of inflammatory heart disease (myocarditis) is higher among men. Proinflammatory cytokines are critical for the development of myocarditis. Recent evidence suggests a link between infections, innate Toll-like receptor (TLR) signaling and increased heart disease. Adjuvants are known to induce inflammatory diseases such as experimental autoimmune myocarditis (EAM) in mice, although the precise mechanisms are unclear. Adjuvants work during the innate immune response to increase proinflammatory cytokines, such as interleukin (IL)-1 and IL- 12, resulting in a T helper (Th)1-type inflammatory response. Recent studies by the PI suggest that coxsackievirus B3 (CVB3) infection induces inflammatory heart disease in mice by mechanisms similar to adjuvant in EAM. We show that TLR4 deficient mice have significantly reduced acute myocarditis and IL-1 levels in the heart and increased levels of the Th1 inhibitory receptor Tim-3, indicating that TLR4 signaling reduces Tim-3 expression during innate immunity resulting in increased inflammation in the heart. Blocking Tim-3 during the innate immune response to CVB3 infection increases TLR4 expression on antigen presenting cells (ARC), indicating that Tim-3 signaling reduces TLR4 expression on ARC following CVB3 infection. Our results demonstrate that cross-talk between TLR4 and Tim-3 signaling regulates the severity of inflammation following viral infection. These results provide a mechanism for how viral infections act similar to adjuvants during the innate immune response to increase acute and chronic myocarditis in males. In this proposal we will examine the following questions using the mouse model of CVB3-induced myocarditis. Aim 1) Is increased myocarditis in males following CVB3 infection mediated specifically by TLR4, or are other TLR signaling pathways involved? Aim 2) Do proinflammatory cytokines (e.g. IL-1) increase myocarditis using mechanisms similar to virus (i.e. increase TLR4 and decrease Tim-3)? Aim 3) Is active viral infection necessary for the development of myocarditis? Do adjuvants increase inflammation using the same mechanisms as virus? Relevance to Public Health: Heart disease is the leading cause of death in the US. This proposal will examine the adjuvant effect of viral infection during innate immunity on the development of heart disease.